Stage-dependent differences in RNA composition and content affect the outcome of expression profiling in roach (Rutilus rutilus) ovary.

The influence of changing composition and content of RNA on the results of expression profiling was studied in the group-synchronous ovaries of roach (Rutilus rutilus) over the course of their maturation. The highest yield of total RNA was detected in the primary growth and early cortical alveolus stages. The total RNA yield gradually decreased through the late cortical alveolus and late vitellogenic stages. In the primary growth and early cortical alveolus stages, total RNA was characterized by a low percentage of 18S and 28S rRNA and a high percentage of smaller-sized RNAs (tRNA, 5S and 5.8S rRNA), whereas 18S and 28S rRNA had increased by the late cortical alveolus stage and dominated by the late vitellogenic stage. The ratio of mRNA to total RNA was highest at the primary growth stage but decreased significantly in later ovarian stages. When total RNA was used for reverse transcription (RT), the shift in the mRNA/total RNA ratio influenced the results of qPCR expression profiling of several commonly used reference genes (ribosomal protein L8, elongation factor-1α, RNA polymerase-subunit B5, and β2-microglobulin) and of two target genes, gonad-type aromatase (cyp19a1a) and follistatin (fst). We conclude that the expression of target genes should be related to the mRNA pool using the same input of either mRNA to RT or cDNA to qPCR. Furthermore, gene expression was related to tissue-specific RNA yield per body mass (RNA yield x ovary mass x body mass⁻¹) thereby reflecting the massive increase in the size and cellular composition of the ovary during the reproductive cycle.